PROJECT SUMMARY Integrin-based focal adhesions (FA) function as mechano-active complexes. Despite significant progress in defining biochemical interactions driving FA assembly and signaling, very little is known about how FAs sense and transmit force and how these forces are integrated into biochemical signals. The objective of this project is to elucidate how forces at FAs are converted into biochemical signals. Our central hypothesis is that the local force balance between ECM-integrin forces and CSK tension at individual FAs regulates FAK phosphorylation levels via vinculin and these force-dependent signals from individual FAs are spatially integrated within the cell to trigger YAP nuclear localization. We will test our central hypothesis by addressing 3 key questions: Q1. Do changes in adhesive force and CSK tension that perturb the local force balance at individual FAs regulate FAK phosphorylation and YAP nuclear localization? We will analyze traction force, vinculin recruitment, and FAK phosphorylation at individual FAs and YAP nuclear localization for fibroblasts and human mesenchymal stem cells cultured on microfabricated post-array-detectors (mPADs). We will evaluate these force-signaling responses on patterned mPADs with different ECM density and elastic moduli and in the presence of contractility modulators to perturb the local force balance between ECM-integrin forces and CSK tension in order to examine how this force balance regulates local FAK phosphorylation and YAP nuclear localization. We will next map spatiotemporal relationships among force, FAK phosphorylation, and YAP nuclear localization in live cells expressing a FRET-based FAK biosensor and fluorescent proteins on mPADs presenting caged RGD peptide. The caged RGD will be activated with UV light to precisely trigger FA assembly in prescribed spatial patterns while monitoring force, FAK phosphorylation, and YAP nuclear localization. Q2. Does FAK regulate mechanosensing at individual FAs and YAP nuclear localization? We will use FAK-null cells expressing wild-type and mutant FAKs to decipher the role of FAK and its Y397 scaffolding and talin- and paxillin-binding sites on mechanosensing. Q3. Does vinculin modulate force-dependent FAK phosphorylation and YAP nuclear localization? We will use vinculin-null cells expressing wild-type and mutant vinculins to analyze how vinculin and its auto-inhibited head- tail conformation modulate force-FAK phosphorylation coupling at individual FAs and YAP nuclear localization. This research will generate new insights into how FAs sense force and how these forces are integrated into biochemical signals. This research will provide a framework to understand cell-ECM mechanotransduction events as well as fundamental principles to design mechanoresponses in cell-biomaterial interactions.